7B2 Knockout transgenic animals as models of endocrine disease

ABSTRACT

In general, the invention features methods and uses for transposon-mediated gene targeting which greatly enhance the insertion and detection of desired genes in genomic exons by homologous recombination. The invention also features transgenic non-human mammals, and eukaryotic cells, wherein a gene encoding 7B2 protein is modified, as well as nucleic acid vectors capable of undergoing homologous recombination with an endogenous 7B2 gene in a cell. The invention also features transgenic non-human mammals as models of endocrine disorders, as well as methods for diagnosing and treating patients with endocrine disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/264,576, filed Mar. 8, 1999, now U.S. Pat. No. 6,548,736, which is acontinuation-in-part of U.S. patent application Ser. No. 09/089,940,filed Jun. 8, 1998, now U.S. Pat. No. 6,504,081, which claims thebenefit of U.S. Provisional Application No. 60/049,523, filed Jun. 13,1997. The specification of U.S. patent application Ser. No. 09/264,576is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The invention relates to gene targeting.

Gene targeting is a process whereby a specific gene, or a fragment ofthat gene, is altered. This alteration of the targeted gene may resultin a change in the level of RNA or protein that is encoded by that gene,or the alteration may result in the targeted gene encoding a differentRNA or protein than the untargeted gene. The targeted gene may bestudied in the context of a cell, or, more preferably, in the context ofa transgenic animal.

Transgenic animals are among the most useful research tools in thebiological sciences. These animals have a heterologous (i.e., foreign)gene, or gene fragment, incorporated into their genome that is passed onto their offspring. Although there are several methods of producingtransgenic animals, the most widely used is microinjection of DNA intosingle cell embryos. These embryos are then transferred intopseudopregnant recipient foster mothers. The offspring are then screenedfor the presence of the new gene, or gene fragment. Potentialapplications for transgenic animals include discovering the geneticbasis of human and animal diseases, generating disease resistance inhumans and animals, gene therapy, drug testing, and production ofimproved agricultural livestock.

SUMMARY OF THE INVENTION

In general, the invention features methods and uses fortransposon-mediated gene targeting which greatly enhance the insertionand detection of desired genes in genomic exons by homologousrecombination. The invention also features diagnostic methods forendocrine disorders, as well as methods and reagents for treatingendocrine disorders.

In a first aspect, the invention provides a method for targetingheterologous DNA to integrate into an exon of a eukaryotic cell. Themethod includes, first, generating a pool of bacteria containingplasmids into which have been randomly integrated a transposon includingheterologous DNA; second, isolating from the pool a bacterium whichcontains a plasmid into which the transposon is integrated into a copyof the exon on the plasmid by assessing PCR amplification productsgenerated from the pool using primers specific for the exon; third,introducing the plasmid of the bacteria into the cell under conditionsthat promote homologous recombination; and, fourth, screening genomicDNA of the cell for integration of the heterologous DNA into the exon ofthe cell.

In one embodiment of the first aspect of the invention, the transposonbears at its extremities recognition sequences of a first rare-cuttingrestriction endonuclease that is absent in the exon. In anotherembodiment, the heterologous DNA, or portion thereof, encodes aselectable marker protein. The heterologous DNA, or portion thereof, mayadditionally encode a second protein, or polypeptide fragment thereof.In another embodiment, the marker protein is a prokaryotic selectablemarker protein, which may be replaced by a eukaryotic selectable markerprotein via the recognition sequences of the first rare-cuttingrestriction endonuclease. The prokaryotic selectable marker protein maybe additionally replaced with DNA, or a portion thereof, encoding asecond protein, or polypeptide fragment thereof.

In another embodiment of this aspect, the exon copy or portion thereofhas at its borders destroyed recognition sequences of a secondrare-cutting restriction endonuclease. In another embodiment, thegenomic DNA is digested with the second rare-cutting restrictionendonuclease. In yet another embodiment, the screening is carried out bySouthern blot analysis of the genomic DNA with a detectable probespecific for the exon, or with a detectable probe external to the exon.The screening may also be carried out by PCR amplification of thegenomic DNA with primers specific for the exon, or with primers externalto, but surrounding the exon such that the PCR product includes theexon.

In a preferred embodiment of the first aspect of the invention, theinsertion of the heterologous DNA into the exon results in a reducedlevel of expression of the protein encoded by the gene of the exon. Theinsertion of the heterologous DNA into the exon may also result in theexpression of a truncated protein encoded by the gene of the exon,expression of a fusion protein encoded by the gene of the exon and theheterologous DNA, or portion thereof, or expression of a product, whichmay be a fusion protein, encoded by the heterologous DNA, or portionthereof.

In a second aspect, the invention provides a method for making atransgenic, non-human vertebrate animal containing heterologous DNA byfirst producing an embryonal cell of the non-human vertebrate animalwith a targeted exon by first, generating a pool of bacteria containingplasmids into which have been randomly integrated a transposon includingheterologous DNA; second, isolating from the pool a bacterium whichcontains a plasmid into which the transposon is integrated into a copyof the exon on the plasmid by assessing PCR amplification productsgenerated from the pool using primers specific for the exon; third,introducing the plasmid of the bacteria into the embryonal cells underconditions that promote homologous recombination; and fourth, screeninggenomic DNA of the embryonal cells to identify an embryonal cell inwhich there has occurred integration of the heterologous DNA into theexon. The identified embryonal cell is then grown to generate thetransgenic animal.

In one embodiment of the second aspect of the invention, the transposonbears at its extremities recognition sequences of a first rare-cuttingrestriction endonuclease that are absent in the exon. In anotherembodiment, the heterologous DNA, or portion thereof, encodes aselectable marker protein. The heterologous DNA, or portion thereof,additionally encodes a second protein, or polypeptide fragment thereof.

In another embodiment, the marker protein is a prokaryotic selectablemarker protein which may be replaced by a eukaryotic selectable markerprotein via the recognition sequences of the first rare-cuttingrestriction endonuclease. In another embodiment, the prokaryoticselectable marker protein is additionally replaced with DNA, or aportion thereof, encoding a second protein, or polypeptide fragmentthereof.

In another embodiment, the exon copy or portion thereof has at itsborders destroyed recognition sequences of a second rare cuttingrestriction endonuclease. Genomic DNA may be digested with the secondrare-cutting restriction endonuclease. In another embodiment, thescreening is carried out by Southern blot analysis of the genomic DNAwith a detectable probe specific for the exon, or with a detectableprobe external to the exon. The screening may also be carried out by PCRamplification of the genomic DNA with primers specific for the exon, orwith primers external to, but surrounding the exon such that the PCRproduct includes the exon.

In a preferred embodiment of this aspect of the invention, the animalexpresses a reduced level of the protein encoded by the gene of theexon. In another embodiment, the animal expresses a truncated proteinencoded by the gene of the exon. In another embodiment, the animalexpresses a fusion protein product encoded by the gene of the exon andthe heterologous DNA, or portion thereof. In another embodiment, theanimal expresses a product, which may be a fusion protein, encoded bythe heterologous DNA, or portion thereof.

In a third aspect, the invention features a transposon that includes aselectable marker cassette including the selectable marker operablylinked to a promoter, or hybrid thereof, capable of expressing themarker in both eukaryotic and prokaryotic cells. In a preferredembodiment of this aspect of the invention, the selectable marker isboth a prokaryotic and eukaryotic selectable marker. In anotherembodiment of this aspect of the invention, the cassette is flanked bythe recognition sequences of one or more rare-cutting restrictionendonucleases. Most preferably, the transposon of this aspect of theinvention is used to integrate a targeted gene, or exon thereof, on aplasmid.

In a fourth aspect, the invention features a eukaryotic cell containingan endogenous exon into which there is integrated a transposon includingDNA encoding a selectable marker.

In a fifth aspect, the invention provides a method for making atransgenic non-human vertebrate animal by providing an embryonal cell ofthe non-human vertebrate animal that includes an endogenous exon intowhich there is integrated a transposon including DNA encoding aselectable marker, and then growing the cell to produce the transgenicanimal.

The invention also features a novel transgenic animal with a geneticallyengineered modification in the gene encoding the 7B2 protein. In a sixthaspect, the invention features a transgenic non-human mammal, wherein agene encoding 7B2 protein is modified resulting in reduced 7B2 proteinactivity. In preferred embodiments of this aspect, the transgenicnon-human mammal is homozygous for the modified gene and is a mouse. Inother preferred embodiments, the gene encoding 7B2 protein is modifiedby disruption, and the transgenic non-human animal has reduced 7B2protein activity, preferably as manifested, e.g., by decreased amount ofmature form PC2 or decreased PC2 protein activity.

In other preferred embodiments of the sixth aspect, the non-humantransgenic mammal is a model of endocrine disease, preferably, theendocrine disease is manifested as a symptom related to Cushing'sdisease, for example, the mammal has increased plasma ACTH, increasedserum corticosterone, or increased distribution of fat in the torso,upper abdomen, or neck.

In further embodiments of the sixth aspect of the invention, thetransgenic non-human mammal has reduced conversion of pro-glucagon,pro-insulin, or pro-enkephalin to mature form. In yet anotherembodiment, the transgenic non-human mammal is heterozygous for the genemodification.

In a seventh aspect, the invention features a nucleic acid vectorcomprising nucleic acid capable of undergoing homologous recombinationwith an endogenous 7B2 gene in a cell, wherein the homologousrecombination results in a modification of the 7B2 gene resulting indecreased 7B2 protein activity in the cell. In a preferred embodiment ofthe seventh aspect, the modification of the 7B2 gene is a disruption inthe coding sequence of the endogenous 7B2 gene.

The eighth aspect of the invention features a eukaryotic cell, whereinthe endogenous gene encoding 7B2 protein is modified, resulting inreduced 7B2 protein activity in the cell. In preferred embodiments, thereduced 7B2 protein activity is manifested, for example, by decreasedamount of mature form PC2 or decreased PC2 protein activity.

In a related aspect, the invention features a eukaryotic cell containingan endogenous 7B2 gene into which there is integrated a transposoncomprising DNA encoding a selectable marker.

Another aspect of the invention features a method for diagnosing amammal for an endocrine disorder, the method comprising determiningwhether 7B2 protein is abnormal, whereby the abnormality indicates thatthe mammal has an endocrine disorder or an increased likelihood ofdeveloping an endocrine disorder. In preferred embodiments, the mammalis a human, the abnormality is reduced 7B2 gene expression, or a nucleicacid mutation in the 7B2-encoding gene, wherein the abnormality resultsin decreased 7B2 protein activity, and the endocrine disorder is ahypercortisolism disorder, preferably Cushing's disease, or ahypoglycemic disorder.

In other preferred embodiments, the abnormality is increased geneexpression, or a nucleic acid mutation in the 7B2-encoding gene, whereinthe abnormality results in increased 7B2 protein activity, and theendocrine disorder is a hypocortisolism disorder, preferably Addison'sdisease, or a hyperglycemic disorder, preferably diabetes.

In other preferred embodiments, expression is measured by assaying theamount of 7B2 polypeptide in the sample, or the amount of 7B2 RNA in thesample.

The tenth aspect of the invention features a method for determiningwhether a compound is potentially useful for treating or alleviating thesymptoms of an endocrine disorder which includes (a) providing a cellincluding a reporter gene operably linked to the promoter from a 7B2gene, (b) contacting the cell with the compound, and (c) measuring theexpression of the reporter gene, such that a change in the level of theexpression in response to the compound indicates that the compound ispotentially useful for treating or alleviating the symptoms of anendocrine disorder.

In a related eleventh aspect, the invention features a method fordetermining whether a compound is potentially useful for treating oralleviating the symptoms of an endocrine disorder, which includes (a)providing a cell that produces a 7B2 protein, (b) contacting the cellwith the compound, and (c) monitoring the activity of the 7B2 protein,such that a change in activity in response to the compound indicatesthat the compound is potentially useful for treating or alleviating thesymptoms of an endocrine disorder.

In a preferred embodiment of the tenth aspect, the 7B2 gene promoter ismammalian, preferably, human or murine. In a preferred embodiment of theeleventh aspect, the 7B2 protein is mammalian, preferably, human ormurine. In other preferred embodiments of the tenth or eleventh aspects,the change is an increase and the endocrine disorder is a hypoglycemicdisorder, or a hypercortisolism/hypercorticosterone disorder, preferablythe disorder is Cushing's disease. In another related embodiment, thechange is a decrease, and the endocrine disorder is a hyperglycemicdisorder, preferably diabetes, or a hypocortisolism/hypocorticosteronedisorder, preferably, the disorder is Addison's disease.

As used herein, by “protein” or “polypeptide” is meant any chain ofamino acids, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation).

By “exon” is meant a region of a gene which includes sequences which areused to encode the amino acid sequence of the gene product.

By “knock-out” is meant an alteration in the nucleic acid sequence thatreduces the biological activity of the polypeptide normally encodedtherefrom by at least 80% compared to the unaltered gene. The alterationmay be an insertion, deletion, frameshift mutation, or missensemutation. Preferably, the alteration is an insertion or deletion, or isa frameshift mutation that creates a stop codon.

By “plasmid” is meant a circular strand of nucleic acid capable ofautosomal replication in plasmid-carrying bacteria. The term includesnucleic acid which may be either DNA or RNA and may be single- ordouble-stranded. The plasmid of the definition may also include thesequences which correspond to a bacterial origin of replication.

By “rare-cutting restriction endonuclease” is meant a restrictionendonuclease whose recognition sequences are located at least 5,000 basepairs apart in the genomic DNA of a mammal. Such restrictionendonucleases include, without limitation, SpeI, NotI, AscI, and PacI.

By “destroyed recognition sequence” is meant the recognition sequence ofa restriction endonuclease which has been destroyed such that thesequence is no longer recognized or cleaved by the restrictionendonuclease. One means of generating a destroyed recognition sequenceis to ligate cleaved ends of recognition sequences from two differentrestriction endonucleases. For example, a SpeI fragment may be ligatedto an XbaI fragment creating ligated DNA having the sequence of 5′ACTAGA 3′ (SEQ. ID NO: 1), which is not recognized by either SpeI orXbaI.

By “operably linked” is meant that a gene and a regulatory sequence areconnected in such a way as to permit expression of the gene productunder the control of the regulatory sequence.

By “selectable marker” is meant a gene product which may be selected foror against using chemical compounds, especially drugs. Selectablemarkers often are enzymes with an ability to metabolize the toxic drugsinto non-lethal products. For example, the pac (puromycin acetyltransferase) gene product can metabolize puromycin, the dhfr geneproduct can metabolize trimethoprim (tmp) and the bla gene product canmetabolize ampicillin (amp). Selectable markers may convert a benigndrug into a toxin. For example, the HSV tk gene product can change itssubstrate, FIAU, into a lethal substance. A preferred selectable markeris one which may be utilized in both prokaryotic and eukaryotic cells.The neo gene, for example, metabolizes and neutralizes the toxic effectsof the prokaryotic drug, kanamycin, as well as the eukaryotic drug,G418.

By “reporter gene” is meant any gene which encodes a product whoseexpression is detectable. A reporter gene product may have one of thefollowing attributes, without restriction: fluorescence (e.g., greenfluorescent protein), enzymatic activity (e.g., lacZ or luciferase), oran ability to be specifically bound by a second molecule (e.g., biotinor an antibody-recognizable epitope).

By “transgenic” is meant any animal which includes a nucleic acidsequence which is inserted by artifice into a cell and becomes a part ofthe genome of the animal that develops from that cell. Such a transgenemay be partly or entirely heterologous to the transgenic animal.Although transgenic mice represent a preferred embodiment of theinvention, other transgenic mammals including, without limitation,transgenic rodents (for example, hamsters, guinea pigs, rabbits, andrats), and transgenic pigs, cattle, sheep, and goats are included in thedefinition.

By “transposon” or “transposable element” is meant a linear strand ofDNA capable of integrating into a second strand of DNA which may belinear or may be a circularized plasmid. Transposons often haveinsertion sequences, or remnants thereof, at their extremities, and areable to integrate into sites within the second strand of DNA selected atrandom, or nearly random. However, only one transposon may integrateinto a second strand of DNA—following insertion of a transposon, thesecond strand of DNA becomes “transposition-incompetent.” Preferredtransposons have a short (e.g., less than ten) base pair repeat ateither end of the linear DNA.

By “protein activity” is meant the functional activity of a givenprotein in a standardized quantity of tissue or cells. The activity of aprotein, as a whole, in such a sample can be modified as a result of achange in the quantity of the given protein present (e.g., as a resultof change in gene expression) or as a result of a change in the functionof each protein molecule present in the sample (e.g., as a result of analteration in amino acid sequence).

By a “mature form” protein is meant the protein form that results fromcomplete, eukaryotic, post-translational processing.

By “endocrine disorder” is meant a disorder affecting the endocrinesystem, resulting in an abnormally increased or reduced levels of anendocrine hormone, or an abnormal response to an endocrine hormone.Endocrine hormones include, without limitation, cortisol,corticosterone, insulin, and glucagon. Exemplary endocrine disordersinclude hypercortisolism (such as Cushing's disease), hypocortisolism(such as Addison's disease), hypoglycemia, and hyperglycemia (such asdiabetes).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram illustrating the cloning of arepresentative SpeI genomic clone into an XbaI site of a targetingvector, resulting in the destruction of the genomic SpeI sites.

FIG. 1B is a schematic diagram representing an in vitro transpositionreaction where the transposon inserts randomly into the targetingvector.

FIG. 1C is a schematic diagram representing a homologous recombinationevent between the transposon-bearing targeting vector and genomic DNA,such that the external SpeI sites are reconstituted.

FIG. 1D is a schematic diagram of a map of the targeting vector CWKO.

FIG. 2A is an agarose gel of PCR amplified DNA from amp/tmp-resistantcolonies resulting from an in vitro transposition reaction.

FIG. 2B is the sequence of the transposition site (SEQ ID NO: 2) in thetargeting vector, indicating that the transposition has taken place intomouse 7B2 exon 3.

FIG. 3A is a schematic diagram representing a homologous recombinationevent between the targeting vector bearing the transposon-inserted 7B2exon 3 DNA and genomic DNA, such that the external SpeI sites arereconstituted and a new SpeI site is added within the neo cassette.

FIG. 3B is a Southern blotting analysis of genomic DNA digested withSpeI from four ES clones probed with labelled transposon DNA.

FIG. 3C is a Southern blotting analysis of genomic DNA digested withSpeI using exonic DNA from mouse neuroendocrine 7B2 as a probe.

FIG. 3D is a Southern blotting analysis of genomic DNA digested withBamHI using exonic DNA from mouse neuroendocrine 7B2 as a probe.

FIG. 4A is a Southern blotting analysis showing all genotypes of miceborn from heterozygote matings, 7B2−/−, 7B2+/−, and 7B2+/+. The mutantallele is 7.6 kb, and the wild type allele is 6 kb.

FIG. 4B is a Northern blotting analysis showing that 7B2−/− mice werenull (i.e., showed no expression) for 7B2 RNA, while 7B2+/+ and 7B2+/−mice did express 7B2 RNA. Equal loading of all lanes is shown bycomparable expression of GAPDH RNA.

FIG. 5A is a photograph showing two four day old 7B2 null mice (incenter) flanked by two four day old wild type mice. The 7B2 null micewere pale and showed significant bruising.

FIG. 5B is a photograph showing 6 week old 7B2 knockout mice (at left)and wild-type mice (at right). Note the marked obesity (e.g., theprominent fat depositions on the back and around the neck) in the 7B2knockout mice.

FIG. 6A is a western blot showing the maturation of propC2 (upper band)to PC2 (lower band). Maturation is significantly impaired in the 7B2knockout mice.

FIG. 6B is a graph showing the activity of PC2 immunopurified frombrains of wild type (closed circles) and 7B2 knockout (open circles)mice. There was a complete absence of PC2 activity in the 7B2 knockoutbrains.

FIG. 7 is a western blotting analysis demonstrating reduced pro-glucagonprocessing in 7B2 knockout mice. Immunoprecipitated glucagon-relatedpeptides were analyzed by gradient SDS-PAGE. Glucagon=pro-glucagon33-61; Glicentin=pro-glucagon 1-69; GRPP-Glu—glicentin-relatedpolypeptide-glucagon, pro-glucagon 1-61; P=pulsed cells; 1C and 3C=1 and3 hour, respectively, pulse chased cells; M=combined chase media.

FIG. 8 is an HPLC profile showing reduced pro-insulin processing in 7B2knockout mice. In wild type mice, pro-insulin I and II were rapidlyconverted to mature form insulin. In contrast, insulin conversion wasslower, with significant accumulations of des-31,32 intermediates (peaksc and d). Peaks are as follows: a=mouse insulin II; b=mouse insulin I;c=des-31,32 mouse proinsulin II; d=des-31,32 mouse proinsulin I; g=mouseproinsulin II; h=mouse proinsulin I.

FIG. 9 is an analysis of immunoreactive enkephalins in acid extractsprepared from 7B2 null (open circles) or wild type (solid circles) mousebrains. Levels of mature enkephalins were reduced in 7B2 knockout mice.

FIG. 10 A-C is a series of charts showing that the 7B2 knockout mice had(A) reduced body weight, (B) hypoglycemia, and (C) hyperproinsulinemia.

FIG. 11 shows the abnormal morphology of the 7B2 knockout pancreas. FIG.11A-B is a pair of charts showing the results of islet cell morphometricanalysis (n=6). The 7B2 knockout mice had increased (A) beta cell mass,and (B) non-beta cell mass. FIG. 11C is a photograph showing generalizedislet cell hyperplasia and abnormal pancreatic morphology in 7B2knockout mice. The 7B2 islets are enlarged, with disordered appearanceof the normally eccentrically-located non-beta cells (stained brown).

FIG. 12 is a photograph showing a typical “buffalo hump” fatdistribution in the 7B2 knockout mice (right panel).

FIG. 13 is a series of photographs of histological analysis of 7B2knockout skin, liver, and spleen. The 7B2 knockout mouse skin isatrophic, hyperkeratotic, and has marked epidermal thinning (FIG. 13B),compared to the wild type skin (FIG. 13A). The 7B2 knockout liver lackslobular architecture, and shows fat vacuolation (FIG. 13D), compared towild type liver (FIG. 13C). The 7B2 knockout mice also exhibit spleniclymphoid atrophy (FIG. 13F) compared to wild type spleen (FIG. 13E).

FIG. 14 is a graph depicting total immunoreactive adrenocorticotropinhormone (ACTH) in each fraction following HPLC analysis of whole mousepituitaries. The 7B2 knockout pituitaries (open circles) showed adramatic increase in ACTH, compared to wild type mice (closed circles),and an absence of corticotropin-like intermediate peptide (CLIP).

FIG. 15 A-C is a series of graphs showing biosynthetic processing ofproopiomelanocortin (POMC) in 7B2 knockout mice (open circles) and wildtype mice (closed circles). Samples were immunoprecipitated withanti-ACTH antiserum and analyzed by SDS-PAGE tube gel system. FIG. 15Arepresents pulse-labeled pituitaries. FIG. 15B represents tissue chasedin unlabeled medium. FIG. 15C represents chase media. The two ACTHfractions represent glycosylated and unglycosylated forms. The 7B2knockout mice showed markedly increased ACTH and reduced a melanocytestimulating hormone (αMSH). FIG. 15D is a diagram showing POMCprocessing. JP=joining peptide.

FIG. 16 is a series of photographs showing reduced αMSH staining in the7B2 knockout mice intermediate pituitary lobes (upper panels;magnification 100×) and the complete lack of ACTH staining in the 7B2knockout anterior lobe (lower panels; magnification 150×).

FIG. 17A is a graph showing elevated plasma corticosterone and ACTHlevels in the 7B2 knockout mice. FIG. 17B is a pair of photographsshowing adrenal cortex (C) expansion in the 7B2 knockout mice (right)compared to wild type mice (left).

DETAILED DESCRIPTION

The present invention describes a novel approach for generatinggene-targeting constructs and generating transgenic animals using theseconstructs, as previously described in application, U.S. Ser. No.09/089,940, herein incorporated by reference. The present inventionfurther describes using this approach to generate a novel transgenicmouse with a 7B2−/− genotype, otherwise known as a 7B2 knockout mouse.

In a simple in vitro reaction using a commercially available transposonand integrals, we have generated random intentional events in aknock-out vector containing thymidine kinase juxtaposed with mousegenomic DNA of interest, the 7B2 gene. Transpositional events wereselected via an antibiotic marker within the transposon. Specific,desired insertions into exonic sequences were subsequently screened forby bacterial colony PCR. Ligation of a neomycin resistance cassette intounique transposon sites within the exon of interest completed thegene-targeting vector, which was shown to undergo homologousrecombination in mouse embryonic stem cells. This approach allowed,within a matter of days, the generation of a completed construct readyfor transfection into embryonic stem cells from a starting genomicclone. This is a general approach that is applicable for intentional“knock-out” and “knock-in” constructs, and allows targeting of differentexons contained within the same genomic clone, independent of convenientrestriction endonuclease recognition sites. Using this technique, anumber of constructs for the same or different genes may be producedsimultaneously.

Transposon-Mediated Generation of Mouse “Knock-Out” Vectors

The conventional technique for generating a “knock-out” mouse entailsplacing a genomic fragment of interest into a vector for fine mapping,followed by cloning of two genomic arms around a neomycin resistancecassette in a vector containing thymidine kinase (Tybulewicz et al.,Cell 61: 1153-1163, 1991). Depending upon skill and luck, thisconventional technique generally requires one to two months for thegeneration of each construct. The single “knock-out” construct is thentransfected into embryonic stem cells, which are subsequently subjectedto positive (using G418) and negative (using FIAU) selection, allowingthe selection of cells which have undergone homologous recombinationwith the knock-out vector. This approach leads to inactivation of thegene of interest (Capecchi, M. R., Trends Genet. 5: 70-76, 1989).

In the transposon-based gene targeting approach of the presentinvention, a genomic fragment containing an exonic sequence of interestwas cloned into a vector containing nucleic acid sequences encodingthymidine kinase and a number of unique restriction endonucleaserecognition sites at the edge of the multiple cloning site. FIGS. 1A-1Drepresent an outline of our transposon-mediated technique for genetargeting.

As a first step shown in FIG. 1A, restriction endonuclease recognitionsites at the edge of a genomic clone of interest were destroyed (in thecase illustrated here, a genomic SpeI fragment was cloned into an XbaIsite of the targeting vector, thereby destroying the genomic SpeIsites). In the second step, as shown on FIG. 1B, a simple in vitrotransposition reaction led to the random integration of a transposoninto the genomic clone. The in vitro transposition reaction was carriedout following the manufacturer's protocol (ABI, Perkin-Elmer Corp.,Norwalk, Conn.). Briefly, 200 ng of transposon, 2 units of integrals, 1g of target plasmid, integrals buffer, and water were incubated at 30°C. for 1 hour. The reaction was stopped by incubation in 0.25 M EDTA, 1%SDS, and 5 g/mL proteinase K for 15 minutes at 65° C. After phenolextraction, the product was precipitated with ammonium acetate andisopropanol, washed in 70% ethanol, and resuspended in 10 L of water. 1L of this product was then electroporated into highly competentbacterial cells, which were then plated on selective medium containing75 g/mL of ampicillin and trimethoprim, since the CWKO vector containsan ampicillin (AMP) resistance gene (bla) and the integrated transposoncontains the trimethoprim (TMP) resistant gene (dhfr). A typicalreaction yielded 100-300 colonies per L, or 1,000-3,000 amp/tmpresistant transposon-bearing colonies from total 10 L transpositionreaction. These colonies became apparent on AMP/TMP agarose plates 12-15hours after electroporation. The transposon bore the recognitionsequences for a number of rare cutting restriction endonucleases at itsextremities, some of which are indicated in FIG. 1B. Thousands ofunique, individual transposition events can be recovered as distinct,doubly-resistant colonies from a typical reaction (Devine and Boeke etal., Nuc. Acid. Res. 22: 3765-3774, 1994). The desired events (i.e.,transpositions into the exon of interest) were discerned via a colonyPCR screen using oligonucleotides homologous to exonic DNA.

Screening by colony PCR was carried out according to the followingprotocol. Single bacterial colonies were dipped into a master mixcontaining 0.4 M primers, 0.2 mM dNTPs, 1×PCR buffer, Taq polymerase,and water. Primers used in this PCR were specific for genomic DNA.Samples were heated to 94° C. for 5 minutes, and then subjected to 30cycles of 45 seconds at 94° C., 30 seconds at 55° C., and 1 minute at72° C. After dipping into the PCR master mix, colonies were touched to amaster plate, which was incubated at 37° C. while PCR and gel analysiswas performed. After completion of the PCR reaction in 2.5 hours, 1.5%agarose gels were loaded with a multichannel pipettor and run out withmarkers, to discern the desired transposition events. Setting up 300 PCRreactions, running the PCR program, and loading and analyzing gels wascompleted in six to eight hours. Colonies found to be positive for thedesired transposition event by PCR were picked from the master plate andproliferated in miniprep format for eight hours. Hence, sticky-endligation of the PGK neo^(c) bpA neo cassette into the targeted exon andsubsequent sequencing of the construct was completed in two days.Completed constructs were sequenced using a standard protocol(Perkin-Elmer Corp., Norwalk, Conn.) and analyzed on an ABI 377automated sequencer.

Ligation of a neomycin resistance cassette into the unique transposonenzyme sites (see FIG. 1B) completed the generation of thegene-targeting construct. Neomycin resistance facilitated the selectionof homologous recombination events based on regaining external enzymesites, as depicted in FIG. 1C, and recombinants were verified bySouthern blot analysis. Only those ES cells which had undergonehomologous recombination regained the original SpeI restrictionendonuclease recognition sites at the edge of the targeted exon atdefined distances from the probe. The desired homologous recombinantsmay then be independently verified by an external probe, if desired.

FIG. 1D is a schematic drawing (not drawn to scale) of the vector,CVVKO, used for this study, wherein all unique sites are listed. Togenerate the CWKO vector, the pSL301 Superlinker plasmid (commerciallyavailable from Invitrogen) was modified in the following manner: HindIIIand NotI sites were filled in with Klenow. A 36 bp hypercleavablerecognition site for PiSceI, which also contains a HindIII site, wasinserted between EcoRI and SalI sites. Note that PiSceI is commerciallyavailable from New England Biolabs (Beverly, Mass.). Oligonucleotideligation created AscI and PmeI sites between the SalI and HindIII sites.Thymidine kinase (TK), isolated from the knock-out vector pPNT(Tybulewicz et al., supra), was blunt-end ligated into a unique MscIsite. Diagnostic digestion verified each unique restriction endonucleaserecognition sequence site listed in FIG. 1D. A genomic fragmentcontaining restriction endonuclease recognition sites listed inparentheses will, when cloned into the cognate unique site in thisvector, destroy those genomic restriction endonuclease sites. Forexample, a SalI-digested fragment cloned into the XhoI site in the CWKOvector will destroy the XhoI site. Restriction endonucleases that areconvenient for linearization of the completed knock-out gene targetingvector are boxed in FIG. 1D. Although other restriction endonucleasesmay be used to linearize the completed targeting vector, it will beunderstood that the site of the recognition sequence of the restrictionendonuclease used to linearize the targeting vector should not belocated in the promoter, coding sequence, or poly A signal associatedwith the targeted gene exon (or inserted transposon therein) or thethymidine kinase encoding sequences (including the promoter and poly Asignal associated with the thymidine kinase-coding sequences).

Targeting of the Murine Neuroendocrine 7B2 Gene

In order to generate a diversity of biologically active peptides,mammals utilize endoproteolysis of biologically inactive polypeptideprecursors. Recently, the prohormone convertase (PC) family of genes hasbeen identified. These serine proteases are involved in the processingof polypeptide hormones such as insulin, glucagon, andproopiomelanocortin (reviewed in Seidah and Chretien, Trends Endocrinol.Metabol. 3: 133-140, 1992; Steiner et al., J. Biol. Chem. 267:23435-23438, 1992). PC2, one of the PCs, interacts with neuroendocrine7B2 in the secretory pathway (Braks and Martins, Cell 78: 263-273,1994).

We chose to use our transposon-based gene targeting approach to targetthe mouse neuroendocrine 7B2 gene. The 7B2 gene was found to be locatedwithin 50 kb of the 3′ end of the formin gene (Wang et al., Genomics 39:303-311, 1997). A 7.5 kb genomic SpeI fragment was isolated from a BAC(commercially available from Genome Systems, St. Louis, Mo.) and clonedinto the XbaI site in the CWKO vector. A simple transposition reaction,entailing incubation and subsequent phenol extraction steps, was thenperformed according to the manufacturer's specifications (Perkin-ElmerCorp., Norwalk, Conn.), as before. Colony PCR reactions were performedusing oligonucleotides homologous to exonic DNA. The primers used were5′-AGTTTTCCCAAGAGGACAGG-3′ (SEQ ID NO: 3) and 5′-TTCTTCCCACGCTGCAGGG-3′(SEQ ID NO: 4), which amplified exon 3 of the mouse 7B2 gene (Braks etal., Eur. J. Biochem 236: 60-67, 1996). The results of the colony PCRreaction indicated that 4 of 288 transposition events were marked byintegration into the exon of interest. FIG. 2A shows a representativepanel of colony PCR products. In clones in which a transposition eventdid not take place in the exon of interest, the endogenous 150 bp exonicband is present, thus indicating transposon integration had taken placeelsewhere in the genomic clone. However, clones in which thetransposition event did take place in the exon of interest showed theexpected up-shift to 1.1 kb. In these clones, the transposon wasinserted into exonic DNA. Such transposition events into the exon ofinterest (i.e., exon 3 of the mouse 7B2 gene) were labelled in FIG. 2Aas 2-53 and 2-70 (note that the transposon is roughly 900 bp). Sequencedata of a clone having a 1.1 kb PCR product, presented in FIG. 2B,confirmed that transposition had indeed taken place into exon 3 of themouse 7B2 gene.

Our transposon-based gene targeting approach has been confirmed to begenerally applicable by generating transpositions into exonic DNA of twoother genomic fragments, which were used for the generation of genetargeting constructs. Since a number of transposition reactions may beperformed in parallel, multiple constructs of different genes can beproduced simultaneously using this procedure.

Homologous Recombination with the Transposon-Mediated Knock-Out Vector

As a final step, we transfected the 7B2 “knock-out” vector generated inthe present study into embryonic stem cells, as described previously(Deng et al., Cell 82: 675-684, 1995). Briefly, 40 g of linearizedtargeting vector was electroporated into embryonic stem (ES) cells andsubjected to positive (G418) and negative (FIAU) selection. Resistant EScell clones were isolated and expanded for genomic DNA isolation. Thisgenomic DNA was subjected to subsequent analyses with Southern blottinganalysis and other standard techniques (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 1994). Linearization of plasmids for transfection into embryonicstem cells was achieved by digestion of the hypercleavable site forPiSceI, which has a 36 base pair recognition site (Gimble and Wang, J.Mol. Biol. 263: 399-402, 1996) and has no reported recognition siteswithin the mouse genome. It is, of course, understood that while PiSceIis ideal to for gene targeting in mice, gene targeting in other animals(e.g., in pigs) is facilitated by use of a restriction endonuclease thathas no or few recognition sites in the porcine genome. FIG. 3A is aschematic diagram showing that homologous recombination led to arestoration of the genomic SpeI sites which were originally destroyed inthe targeting vector (by cloning the SpeI fragment into an XbaI site).Also shown on the schematic in FIG. 3A are genomic SpeI and BamHI sites,as well as an additional SpeI site within the neo cassette. Thepositions of transposon DNA (labelled P1) and exonic DNA (labelled P2)that were used as probes are also indicated in FIG. 3A.

Sixty-three G418-, FIAU-resistant ES clones were obtained, of which twowere proven to have undergone homologous recombination. FIG. 3B showsthat ES clones 50 and 59, which had undergone homologous recombination,had the predicted 5 kb band when using a transposon probe (listed as P1in FIG. 3A). Two other clones did not undergo homologous recombination;ES clone 36 had no detectable band, and ES clone 22 had a band at 4 kb.FIG. 3C shows a genomic Southern blotting analysis using exonic DNA frommouse neuroendocrine 7B2 as a probe (listed as P2 in FIG. 3A). Theprobability of obtaining an insertional event which regained relativelyrare-cutting enzyme sites at precisely the same location on both sidesof the construct is extremely low. Since the neo cassette contained oneSpeI site (see FIG. 3A), the predicted alteration in the genomic locuswas a down-shift from 7.5 kb to a doublet at 5.0 kb and 4.9 kb ingenomic SpeI digested DNA, which was seen in ES clones 50 and 59 in FIG.3C (note that the neo cassette and the transposon make the finaltargeted locus 9.9 kb). This observation was confirmed in FIG. 3D, whichshows a corresponding up-shift from 6 kb to 7.6 kb in a Southern blot ofBamHI digested genomic DNA, using an alternate P2 probe. Note that both“knock-out” (7.6 kb) and endogenous (6 kb) bands were of equalintensity, indicating that the endogenous locus had been targeted.

Analysis of Probability of Homologous Recombination

Using the methods of the invention, the following simple exampleillustrates that, given sufficient numbers of random integration events,a number of desired integrants will almost certainly be isolated. Inthis example, assume that a given genomic clone is 7.5 kb and contains375 bp of exonic sequences (genomic DNA is thought to contain roughly 5%exonic sequences). The chance of one random integration not occurring inthe exonic DNA for this example will then be all non-exonic vector DNAdivided by the total DNA, to the first power. Expressed mathematically,this is ((7.5 kb+2.5 kb-0.375 kb)/(7.5 kb+2.5 kb))¹, i.e.,(9,625/10,000)¹, since the transposon may also insert in 2.5 kb of theknock-out vector which are not taken up by the ampicillin resistancecassette. The chance of 100 random integrations not occurring in theexonic DNA will by extension be (9,625/10,000)¹⁰⁰=2%. As describedherein, 300 colony PCR reactions can be readily performed in under 2hours, and the likelihood of not recovering a desired insert would thenbecome (9,625/10,000)³⁰⁰=0.001%. Thousands of transpositional events perreaction have been routinely obtained, so that the limiting factor isessentially the number of colony PCR reactions one chooses to perform.

Uses for Transposon-Mediated Homologous Recombination

The transposon-mediated gene targeting approach of the invention may begenerally applicable for the generation of insertional knock-outvectors. This technique is rapid, leading from genomic clone to finishedconstruct in a minimum of 4 days, and a number of constructs may begenerated simultaneously. In addition, different exons in the samegenomic clone may be targeted. This can prove useful in proteins inwhich different truncations shed light on the functional significance ofdistinct protein domains. Finally, the generation of knock-in mice,traditionally an arduous task, is greatly simplified by the randomintegration of transposons bearing rare-cutting restriction endonucleaserecognition sequences. Hence, with our technique, cloning any cDNA ofinterest in-frame into a specific genomic locus becomes much lesschallenging and time-consuming.

Transposons for Targeting Genes in Eukaryotic Cells

Certain selectable markers are capable of conveying drug resistance toboth prokaryotic and eukaryotic selection drugs. However, the nucleicacid encoding the selectable marker must be operably linked to apromoter capable of directing expression in both prokaryotic andeukaryotic cells. Such a promoter may be created by fusing a eukaryoticpromoter (e.g., the PGK promoter) with a prokaryotic promoter (e.g., asynthetic EM-7 E. coli promoter). For example, nucleic acid encoding theneo marker protein may be operably linked to the fusion promoter. Aconsensus poly A signal capable of terminating both prokaryotic andeukaryotic transcription may be positioned 3′ to the nucleic acidencoding neo. Employment of a transposon incorporating this modified neocassette will enable the propagation of transposon-integrated CWKOplasmids in bacteria grown in the presence of both ampicillin andkanamycin. Once a plasmid bearing a transposon insertion into a desiredgene, or exon thereof, is identified, the plasmid may be directlylinearized and used to homologously recombine eukaryotic cells, therebybypassing the replacement of a prokaryotic selectable marker with aeukaryotic selectable marker. Resulting homologously recombinedeukaryotic cells are resistant to both FIAU and G418.

Another gene capable of conferring drug resistance in both eukaryoticand prokaryotic cells is the Zeocin™ resistance gene which confersresistance to the drug, Zeocin™. The Zeocin™ drug and Zeocin™ resistancegene are both commercially available from Invitrogen (San Diego,Calif.). The Zeocin™ resistance gene cassette (nucleic acid encoding theZeocin™ resistance marker protein operably linked to a hybrid promoterthat includes the eukaryotic CMV promoter and the bacterial syntheticEM-7 promoter) may be readily removed from the pZeoSV2 vector(Invitrogen, Carlsbad, Calif.) and subcloned into the transposon.Preferably, when the Zeocin™ resistance gene cassette is inserted intothe transposon, it is flanked by rare cutting restriction endonucleaserecognition sequences.

Eukaryotic Cells with One or More Targeted Genes

The utilization of the methods of the invention, as described, willgreatly facilitate the generation of mice with targeted genes. Given therapidity of the tranposon-based generation of targeting vectors, it isunderstood that more than one vector can be produced at the same time.For example, the in vitro transposon reaction may be applied to a murinegenomic library in the CWKO vector. Methods for the generation of such alibrary are well known in the art (see, for example, Ausubel et al.,supra). Murine genomic DNA is also commercially available (from, e.g.,Clontech Laboratories Inc., Palo Alto, Calif.), and may be readilyprepared for insertion into the CWKO vector. Following integration ofthe tranposons, bacterial colonies may be subjected to PCR colonyscreening using primers specific for all desired targeted genes. Forexample, the bacterial colonies may first be screened for transposoninsertion into the mouse neuroendocrine 7B2 gene. Followingidentification of colonies which have targeted 7B2, the remainingcolonies may next be screened for transposon insertion into a secondgene, e.g., actin. Following identification of actin-targeted colonies,the remaining colonies may be screened for transposon insertion into yetanother gene of interest. Since bacteria colony containing plates areeasily duplicated, a genomic library carrying transposon insertions maybe maintained indefinitely in bacteria (with appropriate passaging ofcolonies onto fresh AMP/TMP plates) for future screens for targetedgenes of interest. Likewise, the plasmid DNA from these bacteria may beisolated by standard maxi-prep techniques, and re-transformed intobacteria for expansion when a future screen is desired.

Once a transposon insertion event into a targeted gene is identified, aeukaryotic selectable marker is inserted into rare-cutting restrictionendonuclease recognition sites located on the transposon inserted intothe gene of interest, or an exon thereof. The sites preferably flank thedhfr prokaryotic selectable marker gene. It is understood that anyeukaryotic selectable marker may be utilized (e.g., hygB, pac, hisD,neo). For example, an exon from the murine neuroendocrine 7B2 gene maybe inserted with neo, while an exon from the murine actin gene may beinserted with pac. The targeting vector is then linearized and used tohomologously recombine with chromosomal DNA in eukaryotic cells (e.g.,murine ES cells), which are then treated with FIAU and the drugcorresponding to the transposon-inserted eukaryotic marker. It will beunderstood that linearized vectors may be from different targetingvectors; however the two vectors preferably bear exons inserted with twodifferent eukaryotic selectable markers. For example, both theneo-inserted neuroendocrine 7B2 gene and the pac-inserted actin gene maybe targeted in the same murine ES cell. The ES cells are then subjectedto selection in FIAU, G418, and puromycin.

It is understood that this simultaneous targeting of more than one genemay be utilized for the development of “knock-out mice” (i.e., micelacking the expression of a targeted gene product), “knock-in mice”(i.e., mice expressing a fusion protein or a protein encoded by a geneexogenous to the targeted locus), or mice with a targeted gene such thata truncated gene product is expressed.

Although the use of a genomic library does not allow the destruction ofa restriction endonuclease recognition site flanking the targeted geneexon, homologous recombination events in ES cells may be screened for bySouthern blot alone without the additional screen for restoration of thedestroyed restriction endonuclease recognition site. Should more thanone gene be targeted, Southern blot analysis with probes from both genesmay be utilized. If the genes are of detectably different sizes, bothprobes may be used at the same time.

Eukaryotic Cells with a Targeted Gene which Partially Encodes a FusionProtein

Cells and rodents expressing fusion proteins of proteins tagged withlacZ or GFP (green fluorescent protein) have been utilized for precisedevelopmental expression studies (LeMouellic et al., Proc. Natl. Acad.Sci. USA 87: 4712-4716, 1990; Mansour et al., Proc. Natl. Acad. Sci. USA87: 7688-7692, 1990; Sosa-Pineda et al., Nature 386: 399-402, 1997). Inaddition, fusion proteins of targeted gene products fused to an oncogenehave been used as a model for human cancer translocations (Corral etal., Cell 85: 853-861, 1996; Castilla et al., Cell 87: 687-696, 1996;Yergeau et al., Nature Gen. 15: 303-306, 1997). Utilization of themethods of the invention will greatly facilitate the construction ofsuch transgenic cells and animals. For example, an exogenous gene,encoding, e.g., lacZ, may be fused to a targeted gene at the carboxyterminus of the targeted gene product by subcloning an exon into theCWKO vector. Following identification of a transposon insertion into theexon by the methods described herein, the dhfr gene on the transposonmay be replaced with nucleic acid from a desired exogenous gene (e.g.,lacZ or an oncogene) separated from the nucleic acid encoding aeukaryotic selectable marker (e.g., the PGK neo^(c) bpA neo cassette)with a stop codon such that the inserted nucleic acid from the desiredexogenous gene is in frame with and adjacent to the exon. The insertednucleic acid encoding a eukaryotic selectable marker is, thus, locatedat the extreme carboxy-terminal end of the fusion protein. It isunderstood that should the exogenous gene be fused to the targeted geneat the amino terminus of the targeted gene, the heterologous DNAinserted into the transposon integrated into the targeted exon includesthe entire neo cassette located 5′ to a promoter operably linked to theexogenous gene of choice. The exogenous gene is inserted into thetransposon such that it is in frame with and 5′ adjacent to the exon ofthe targeted gene. The targeting vector is next homologously recombinedinto eukaryotic cells. Genomic DNA of FIAU and G418 resistant clones maythen be screened for restoration of the destroyed restrictionendonuclease sites on the exon and by Southern blot analysis with aprobe corresponding to exonic DNA.

Eukaryotic Cells with a Targeted Gene Encoding a Truncated Protein

Cells expressing truncated proteins are useful in analysing the roles ofspecific domains of proteins in the biological functions of the fulllength proteins. For example, should the targeted gene produce a productwhose two functional domains are separated by the amino acid residuesencoded by the fourth exon, the fourth exon may be subcloned into theCWKO vector and subjected to an in vitro transposition reaction. Once atransposon has inserted into exon 4, the dhfr gene on the transposon maybe replaced with a eukaryotic selectable marker cassette (e.g., PGKneo^(c) bpA) plus addition sequences. For a C-terminal truncatedtargeted gene product, the selectable marker cassette bears additionalsequences 5′ to the PGK promoter. These 5′ sequences include stop codonsin all three frames, followed by a poly A signal, such that exon 4transcription terminates prior to the initiation of transcription of theselectable marker cassette. Should a N-terminal truncated targeted geneproduct be desired, the additional sequences are 3′ to the selectablemarker cassette and include a promoter operably linked to an initiatorcodon that is in frame with exon 4 such that the truncated protein isexpressed from DNA (i.e., exon 4) located 3′ to the transposon insertionsite. A cell bearing the truncated protein may then be detected by therestoration of the destroyed restriction endonuclease sites flankingexon 4, and by Southern blot analysis using exon 4 DNA as a probe.

Eukaryotic Cells with Nucleic Acid Encoding a Protein Introduced intothe Locus of a Targeted Gene

Several recent papers have indicated the power of “knock-in” technologyin analyses of the functional complementation between related genes(Hanks et al., Science 269: 679-682, 1995). The methods of the presentinvention will greatly facilitate the rapidity with which these mice maybe generated. Generation of “knock-in” cells and mice is accomplished bydesigning a targeting vector in which the prokaryotic selectable markergene (e.g., dhfr) located on the transposon is replaced with nucleicacid (e.g., genomic DNA or, more preferably, cDNA) encoding a secondprotein together with a eukaryotic selectable marker cassette.Homologous recombination of this targeting vector with the chromosomalDNA in eukaryotic cells (preferably, ES cells) is accomplished byscreening genomic DNA for restoration of destroyed restrictionendonuclease recognition sequences flanking the targeted exon and bySouthern blot analysis with probes corresponding to the targeted exon.

Utilization of Eukaryotic Cells with Targeted Genes

A eukaryotic cell with one or more targeted genes allows the analysis ofthe effects of the targeted gene in the cell. For example, a terminallydifferentiated CD8⁺ T cell may have a targeted disruption of the lckgene, such that no lck protein is expressed. Although the targeted geneis present on only one chromosome, lack of lck expression may beaccomplished nevertheless, since most terminally differentiated cellsare functionally hemizygous. This cell may be used for the functionalanalysis of antigen responsiveness in the absence of lck, and todetermine if other endogenous protein tyrosine kinases can compensatefor the lack of lck. Similarly, this cell may express a fusion proteinof lck fused to the fluorescent marker, green fluorescent protein (GFP).The subcellular localization of the lck protein may then be assessedduring the various biological responses of the protein.

A murine ES cell bearing targeted gene(s) may be used to generateheterozygous and homozygous mice using standard techniques (Tybulewiczet al., supra; Capecchi, supra). Hence, depending upon the type ofdisruption in the targeted gene, mice with no expression of the targetedgene, expression of a fusion protein partially encoded by the targetedgene, or expression of a different gene product from the targeted genelocus may be generated. Analysis of the effects on the disruptedtargeted genes may then be assessed on an organismal level. In addition,murine embryonic fibroblast cells (MEFs) may be derived from murine EScells or transgenic mice according to standard procedures (Deng et al.,Cell 82: 675-684, 1995), and may allow more detailed studies in cellculture.

7B2 Knock-Out Mice Genotype

After electroporation of the 7B2 gene-targeting construct into embryonicstem (ES) cells, several ES clones were shown to have undergonehomologous recombination. Two of these were injected into blastocysts,chimeric mice were derived, and germline transmission was shown tooccur. Chimeric mice were mated to 129Svev strain females to place thetargeted 7B2 gene on a pure genetic background. All of our mice 7B2“knockout” mice, that is, mice with 7B2−/− genotypes, were derived fromthese two independent ES clones. No difference was noticed between thetwo independent mouse lines.

Genotyping of heterozygotic matings indicated offspring of all possiblegenotypes (see FIG. 4A). In order to analyze 7B2 RNA levels, total RNAwas extracted from whole brains of 7B2+/+(wild type) and 7B2−/−(knockout) mice, and subjected to Northern blotting analysis using the7B2 exon 2 DNA as a probe according to standard techniques (see, e.g.,Ausubel et al., supra). As shown in FIG. 4B, 7B2 knockout mice werefound to lack detectable 7B2 RNA transcripts.

General Knockout Phenotype

From an early age, the 7B2 knockout mice exhibited clear clinicalabnormalities. At four days of age, 7B2 knockout mice (two are shown inthe middle of FIG. 5A; two wild type mice are flanking them) wereobserved to be pale and ecchymotic (note especially the severe bruisingof the left 7B2 knock-out mouse). Many 7B2 null mice suffered fromsignificant bleeding into the abdomen. Only 11% of 7B2 null micesurvived to weaning, and 7B2 null mice were often very severely runted,with parchment-like. Despite this significant runting, however, those7B2 null mice which did survive weaning actually became obese afterweaning, with a prominent fat deposition on the back and around the neck(six week old 7B2 null and wild type mice are shown in FIG. 5B).

PC2 Expression Form and Activity

Several studies have indicated that PC2 activity might be dependent upon7B2 function (Braks and Martens, Cell 78: 263-273, 1994; Braks andMartens, FEBS Lett. 371: 154-158, 1995; Braks et al., Eur. J. Biochem.238: 505-510, 1996; Zhu et al., J. Biol. Chem. 271: 23582-23587, 1996;Zhu et al., Proc. Natl. Acad. Sci. USA 93: 4919-4924, 1996). OurSDS-PAGE analysis of PC2 protein expression forms in brain (where PC2 isexpressed at high levels), revealed that the maturation of propC2 tomature PC2 was severely inhibited in 7B2 knock-out mice (FIG. 6A, rightlane, n=3).

Fluorometric PC2 activity assays were performed on PC2 samples whichwere immunopurified from whole brain protein extracts using anti-PC2polyclonal antibody. The PC2 activity assays were performed by standardprocedures (see, e.g., Lindberg et al., Biochemistry 34: 5486-5493,1995; Zhu et al., J. Biol. Chem. 271: 23582-23587, 1996, hereinincorporated by reference). Briefly, mouse brains were homogenized in anon-denaturing detergent (e.g., not SDS) and TBS. After centrifugationto obtain soluble proteins, PC2 antibody was added (pre-bound to ProteinA beads). This mixture was incubated for 4 hours at 4° C. The beads werethen pelleted by centrifugation and washed in TBST.

The fluorometric assay was based on PC2-mediated liberation ofaminomethylcoumarin (AMC) using the fluorogenic substrate,pGlu-Arg-Thr-Lys-Arg-AMC (commercially available from PeptidesInternational, Lexington, Ky.), as previously described (Zhu andLindberg, J. Cell. Biol. 129: 1641-1650, 1995, herein incorporated byreference). The fluorescent standard AMC (commercially available fromPeninsula Laboratories Inc., Belmont, Calif.) was used to calibrate thefluorometer. As shown in FIG. 6B, 7B2−/− mice completely lacked PC2activity, indicating that PC2 activity is dependent upon 7B2.

Prohormone Processing Abnormalities

Consistent with the lack of PC2 activity, the 7B2 knockout miceexhibited abnormal production and processing of glucagon, insulin, andenkephalins, all of which are hormones dependent upon PC2 for normalprocessing.

With regard to proglucagon processing, pancreatic islets werepulse-chase labeled, and, subsequent to immunoprecipitation,glucagon-related proteins were subjected to SDS-PAGE. As shown in FIG.7, 7B2 knockout islets displayed minimal conversion of pro-glucagon tomature glucagon, with the majority of glucagon-related proteinsremaining as unconverted pro-glucagon and only small amounts ofintermediate glucagon cleavage products. In contrast, wild type isletsdisplayed a rapid and almost complete conversion of pro-glucagon toglucagon in wild type islets.

To assess insulin processing, pancreatic islets were similarly labeled.In wild type islets (FIG. 8, left panel), mouse proinsulin I and II wasrapidly converted to insulin. In contrast, insulin maturation wassignificantly delayed in knock-out islets, and was accompanied by thegeneration of increased amounts of 20 des 31,32 proinsulin intermediatematerial (peaks c and d in FIG. 8, right lower panel). This intermediateof proinsulin is produced by PC1 and PC3, which exhibits a cleavagepreference for the B chain-C peptide junction.

The levels of two mature enkephalins were also dramatically reduced inthe 7B2 knock-out brains (FIG. 9).

Pancreatic Abnormalities

The 7B2 knockout mice had lower blood glucose levels and elevated levelsof circulating insulin-related protein, as shown in FIG. 10. Inaddition, the knockout mice also had lower body weight (FIG. 10). Bloodwas analyzed using a standard glucometer, and plasma insulin wasmeasured by radioimmunoassay.

The metabolic disturbances observed in the 7B2 null mice wereaccompanied by morphological effects on the pancreas. For morphometricanalysis, mouse tissue (e.g., pancreas) was removed and fixed in OptimalFix (American Histology Reagent, Lodi, Calif.), blocked in paraffin,sectioned at 10 m, and stained with hematoxylin and eosin, according tostandard techniques. As shown in FIGS. 11A and 11B, pancreatic beta andnon-beta islet masses were significantly increased in 7B2 knockout mice.FIG. 11C shows photographs at 20× magnification of representativepancreas specimens from 7B2 knockout (KO) and wild-type (WT) mice atfive weeks of age, indicating both the increased size and markedlyabnormal morphology of islets in the pancreas of 7B2 null mice. Theislets in the 7B2 knockout were hyperplastic and had disorderedarchitecture, with disruption of the normal eccentric location ofnon-beta cells.

7B2 Knockout Versus PC2 Knockout

Given the 7B2 knockout mice were found to lack PC2 activity, PC2activity is dependent upon 7B2 function. Consistent with this 7B2-PC2inter-relationship, the 7B2 knockout mice were found to exhibitphenotypic characteristics similar to the those reported for PC2knockouts. For example, like the 7B2 knockout mice of the presentinvention, the PC2 knockout mice are hypoglycemic andhyperproinsulinemic, with generalized islet cell expansion, alteredislet cell morphology, and depressed levels of bioactive peptides suchas mature enkephalins and glucagon (Furuta, 1997, Johanning et al., 1998Rouille et al., 1994, Rouille et al., 1997). The islet cell changesdiffered in the 7B2 knockout in that the beta cell mass was alsoincreased, possibly due to elevated corticosterone (see below) or anindirect steroid-induced insulin resistance.

More surprisingly, however, the 7B2 knockout mice exhibited additionalphenotypic abnormalities, such as Cushing's disease-like abnormalities,which were not present in the PC2 knockouts. These additionalcharacteristics provide evidence that 7B2 has additional actions whichare independent of PC2.

Cushing's Disease-Related Abnormalities

All of the 7B2 knockout mice surviving past four weeks of age exhibitedan abnormal pattern of fat distribution around the back of theneck—otherwise known as a “buffalo hump” (see FIG. 12). This type of fatdistribution pattern is commonly observed in humans suffering fromCushing's disease, a disease associated with hypersecretion of cortisolby the adrenal cortex (or over production of other similar steroidhormones, such as hydrocortisone, prednisone, methyl-prednisolone, ordexamethasone). The hypersecretion of cortisol can result from a generalhyperplasia of one of both adrenal cortices, which may, in turn, becaused by increased secretion of adrenocorticotropin hormone (ACTH) bythe anterior pituitary.

Further histological analysis of the 7B2 knockout mice revealed othersymptoms consistent with Cushing's disease, such as the following: theskin of 7B2 knockout mice (FIG. 13B, 20×) showed marked thinning andepidermal hyperkeratosis, as well as dermal atrophy, as compared to wildtype mice (FIG. 13A, 20×); in the 7B2 knockouts' livers, the normallobular liver architecture was destroyed and severe fat vacuolation waspresent (FIG. 13D knockout versus FIG. 13C wild type, 20×magnification); the spleens in the 7B2 knockout mice were roughlyone-fifth the wild type size and showed abnormal architecture and ageneralized myeloid immaturity (FIG. 13F knockout versus FIG. 13E wildtype, 5× magnification).

Turning to ACTH, analysis of total pituitary ACTH revealed that the 7B2knockout mice had a 10-20 fold increase in intact ACTH, and nodetectable corticotropin-like intermediate peptide (CLIP), an ACTHcleavage product (FIG. 14). Similarly, biosynthetic studies ofprocessing of ACTH precursor proopiomelanocortin (POMC) in isolatedwhole pituitaries showed that 7B2 knockout mice had elevated productionof intact ACTH, with minimal conversion to a melanocyte stimulatinghormone (αMSH) (FIG. 15).

For measurement of pituitary ACTH, pituitaries from 7B2 knockout orwild-type mice were homogenized in ice cold 1N acetic acid bysonication. Following microcentrifugation of the homogenates, aliquotswere then injected into a high pressure gel permeation chromatograph andrun in 32% acetonitrile plus 0.1% trifluoroacetic acid. Fractions werethen assayed using ACTH-IR peptides directed against residues 11-17 ofACTH.

Both the anterior and the intermediate lobes of the pituitary synthesizethe ACTH precursor, POMC. In the anterior lobe of PC2 or 7B2 knockoutmice, ACTH levels are unaffected because cleavage of POMC into fulllength ACTH (the end product in this lobe) occurs primarily through theaction of PC1 and PC3 rather than PC2 (Bloomquist et al, 1991; Benjannetet al, 1991; Thomas et al, 1991; Zhou et al, 1993). However, ACTH levelsare affected in the intermediate lobe of PC2 or 7B2 knockout micebecause PCT is highly expressed and cleaves ACTH into thenon-corticotropic peptides MSH and CLIP (Zhou et al, 1993; Benjannet etal, 1991; Thomas et al, 1991).

FIG. 16 (a versus b) shows that the intermediate lobe of 7B2 knockoutmice exhibit reduced MSH cleavage, suggesting an accumulation of ACTH.(In the intermediate lobe, ACTH antiserum would cross-react with CLIPand would interfere with assessment of ACTH. Therefore, ACTH processingwas measured using MSH-specific antiserum.) Thus, ourimmuno-histochemical studies suggest that the increased pituitary ACTHin the intermediate lobe causes the increased total pituitary ACTH. Incontrast, there was a marked reduction in anterior lobe ACTH staining in7B2 knockout mice (FIG. 16; panels c and d).

In conjunction with increased pituitary ACTH, plasma ACTH, as well asserum corticosterone levels, were increased in the 7B2 knockout mice(FIG. 17A). Corticosterone assays were performed on serum as describedpreviously (Meiner et al., Proc. Natl. Acad. Sci. USA 93: 14041-14046,1996). In addition, the adrenal cortex, which produces corticosterone,was greatly expanded in 7B2 null mice (FIG. 17B).

In summary, the 7B2 knockout mice contrast sharply with the PC2knockouts in that the 7B2 knockout mice exhibit high levels of plasmaACTH and go on to develop symptoms related to severe pituitary Cushing'sdisease, surviving at most to 9 weeks after birth. The adrenal corticalhyperplasia observed in these animals, a consequence of continuoustrophic stimulation by increased circulating ACTH, results in highplasma levels of corticosterone in the 7B2 knockout. Elevated levels ofplasma steroids cause a number of phenotypic changes observed in thismouse, such as atrophic skin, lipodystrophy, and splenic lymphoidatrophy. The runting of 7B2 mutant pups may also be due to chronicallyincreased circulating corticosterone. None of these changes were notedin reports on PC2 knockout mice; they exhibit no detectable dysfunctionof the pituitary/adrenal axis. These data imply that profounddifferences exist in the 7B2 and PC2-mediated control of intermediatelobe pituitary secretion.

These differences point to important additional functional roles for 7B2not related to PC2-mediated effects. The hypothesis of additional rolesfor 7B2 is strengthened by recent findings that 7B2 is found in brainareas lacking PC2, while the converse has never been observed (Seidel etal, 1998). With respect to the increased secretion of ACTH from thepituitary of 7B2 knockout animals, these novel effects of 7B2 might bedue to developmental changes during maturation of the intermediate lobeaffecting the number and size of melanotrophs, changes in theinnervation of this lobe affecting secretory activity, or effects of 7B2on dopaminergic and GABAergic innervation of the pituitary intermediatelobe.

Other genetic models for pituitary Cushing's syndrome include the D2receptor knockout cited above (Saiardi and Borrelli, 1998) and the CRHtransgenic mouse (Stenzel-Poore et al, 1992). Interestingly, despitepresenting qualitatively similar steroid-induced tissue changes as the7B2 knockout, the CRH transgenic mouse and the D2 receptor knockoutmouse both exhibit a much less severe Cushing's phenotype, with normallifespans. These data highlight the fact that loss of 7B2 expressionaffects pituitary secretory activity in a much more profound manner thanloss of CRH regulation, dopamine receptors, or PC2.

For example, the lack of severe Cushing's syndrome in the dopaminereceptor and transporter knockouts implies that while developmentalalterations in dopaminergic innervation can potentially contribute tothe pathogenesis of this disease, other secretory deficits must also bepresent in the 7B2 knockout which culminate in the exceptionally highcirculating ACTH levels in this animal. In support of this assertion,preliminary results indicate that basal release of intact ACTH isgreatly enhanced in isolated pituitaries of 7B2 knockout animalscompared to controls suggesting that isolated pituitaries retain theproperty of hypersecretion even when removed from direct dopaminergicinfluence. 7B2 may interact with an as-yet undiscovered prohormoneconvertase that is involved in POMC processing.

Further study of our 7B2 knockout mouse will allow detailedcharacterization of 7B2 control of steroidogenesis, either direct orindirect, which is important for a proper understanding of both normalhuman physiology, as well as hypercortisolic disease states (e.g.,Cushing's disease), hypocortisolic disease states (e.g., Addison'sdisease), hypoglycemia, and hyperglycemia (e.g., diabetes).

The Role of 7B2 in Endocrine Disorders

Our unexpected finding that 7B2 knockout mice have Cushing's diseaseallows for the development of methods and reagents to treat or diagnosepatients having (or suspected of having) endocrine disorders. Any cell,tissue, or product of the 7B2 knockout mouse (or the mouse itself),which lacks 7B2 RNA or 7B2 protein may be used as a model forunderstanding endocrine mechanisms. Mice heterozygotic for the 7B2mutation are also useful

These mice are useful for developing methods and reagents for treatingor diagnosing patients having, or suspected of having, ahypercortisolism disorder such as Cushing's disease, or a hypoglycemicdisorder. Furthermore, these mice may find use in the development ofmethods and reagents to treat or diagnose patients having (or suspectedof having) a hypocortisolism disorders, such as Addison's disease, or ahyperglycemic disorder. In accordance with the teachings of theinvention, the sequence of the human 7B2 gene and protein (Braks et al.,Eur. J. Biochem. 236: 60-67, 1996; Martens, G. J., FEBS Lett. 234:160-164, 1988) may be thus manipulated to provide therapeutic reagentsand methods for patients suffering from an endocrine disorder.

The 7B2 knockout mice, as well as the 7B2 heterozygote mice (+/−; seeFIGS. 4A and 4B), provide excellent non-human models for live-animalscreens of any compound (including those compounds isolated using themethods described below) suspected of being useful as a therapeutic totreat or alleviate symptoms in patients suffering from endocrinedisorders such as hypercortisolism disorders (e.g., Cushing's disease),hypocortisolism disorders (e.g., Addison's disease), hypoglycemia, orhyperglycemia (e.g., diabetes). In addition, cell lines derived from 7B2knockout or heterozygote mice could also be used in compound screens.

A therapeutic compound for use in patients having (or suspected ofhaving) a hypercortisolism disorder will alleviate at least one, andpreferably at least two, of the symptoms of these mice. Hence, acompound that can treat or alleviate the disease symptoms ofhypercortisolism disorder, when administered to a 7B2 knockout mouse,will lead to at least one of the following: a restoration of normal PC2activity; a reduction in runting; a restoration of normal skin coloringand a reduction in bruising; a reduction in bleeding into theperitoneum; a reduction of the “buffalo hump;” a reduction in obesity; arestoration of normal pancreatic islet cell mass; a restoration ofnormal blood glucose levels; a restoration of normal plasma insulin andglucagon levels; a restoration of a morphologically normal liver; arestoration of a morphologically normal spleen; a restoration of anormal level of ACTH; a restoration of a normal serum concentration ofcorticosterone; and a restoration of a morphologically normal adrenalcortex.

Diagnostic Methods for Endocrine Disorders

For diagnostic methods, a patient suspected of having or developing anendocrine disorder, such as hypocortisolism, hypercortisolism,hypoglycemia, or hyperglycemia, may be tested for the level ofexpression, or the level of protein activity, of the neuroendocrine 7B2gene. The 7B2 expression level may be measured at the protein or RNAlevel, and may be deemed to be normal or abnormal (e.g., reduced orincreased) by comparison to the level of a control individual. 7B2protein activity level may be measured, for example, by the ability ofthe protein to interact with, and convert, PC2.

A patient suspected of having or developing an endocrine disorder mayalso be screened for a mutation in the gene encoding the 7B2 protein.The mutation can be detected by analyzing genomic DNA, RNA, or mRNA,collected from any tissue. The effect of the mutation could bedetermined by measuring the 7B2 protein amount and/or activity.

Any abnormality in the 7B2 gene resulting in a reduction in the amountor activity of the 7B2 protein is an indication that the patient mayhave, or may be predisposed to develop, a hypercortisolism disorder,such as Cushing's disease, or a hypoglycemic disorder. Conversely, anyabnormality in 7B2 gene expression or gene sequence that results in anincrease in the amount or activity of the 7B2 protein is an indicationthat the patient may have, or may be predisposed to develop, ahypocortisolism disorder, such as Addison's disease, or a hyperglycemicdisorder.

7B2-Targeted Screens: Identifying Therapeutic Candidates for Treating anEndocrine Disorder

For a patient suffering from an endocrine disorder, our discovery allowsfor the development of reagents that may alleviate the disease symptoms.It will be understood that such a patient may or may not show an altered(i.e., abnormal) level of 7B2 protein expression or activity.

7B2 protein, or DNA encoding the 7B2 protein (e.g., the 7B2 gene), maybe administered to neuroendocrine cells or pituitary cells in patientssuffering from a hypercortisolism disorder such as Cushing's disease, orpatients suffering from hypoglycemia Similarly, compounds identified in7B2 screens as increasing the expression of 7B2, or 7B2 proteinactivity, may be administered to patients with a hypercortisolismdisorder or with hypoglycemia. Such compounds could include smallmolecules, nucleic acids or proteins.

In addition, compounds identified in 7B2 screens as reducing 7B2expression or activity, for example, antisense 7B2 nucleic acid, 7B2neutralizing antibody, or 7B2 polypeptide fragments, may be administeredto neuroendocrine cells or pituitary cells in patients suffering from ahypocortisolism disorder such as Addison's disease, or patientssuffering from hyperglycemia.

Preferably, compounds identified in the above described screens modify7B2 expression or protein activity by at least 25%, more preferably byat least 50%, more preferably by 70%, and most preferably by 100%.

Test Compounds

In general, drugs for prevention or treatment of an endocrine disorderwhich function by altering the amount or level of biological activity ofa 7B2 protein are identified from libraries of natural products orsynthetic (or semi-synthetic) extracts or chemical libraries accordingto methods known in the art. Examples of such extracts or compoundsinclude, but are not limited to, plant-, fungal-, prokaryotic- oranimal-based extracts, fermentation broths, and synthetic compounds, aswell as modification of existing compounds. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Libraries of genomic DNA or cDNA may begenerated by standard techniques (see, e.g., Ausubel et al., supra) andare also commercially available (Clontech Laboratories Inc., Palo Alto,Calif.). Nucleic acid libraries used to screen for compounds that alter7B2 gene expression or 7B2 protein activity are not limited to thespecies from which the 7B2 gene or protein is derived. For example, aXenopus cDNA may be found to encode a protein that alters human 7B2 geneexpression or alters human 7B2 protein activity.

Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.

In addition, methods for dereplication (e.g., taxonomic dereplication,biological dereplication, and chemical dereplication, or any combinationthereof) or the elimination of replicates or repeats of materialsalready known for their therapeutic activities for neuroendocrine orpituitary disorders can be employed.

When a crude extract is found to prevent or delay onset of an endocrinedisorder, further fractionation of the positive lead extract isnecessary to isolate chemical constituents responsible for the observedeffect. Thus, the goal of the extraction, fractionation, andpurification process is the characterization and identification of achemical entity within the crude extract having endocrinedisorder-preventative or -palliative activities. The same assaysdescribed herein for the detection of activities in mixtures ofcompounds can be used to purify the active component and to testderivatives thereof. Methods of fractionation and purification of suchheterogenous extracts are known in the art. If desired, compounds shownto be useful agents for treatment are chemically modified according tomethods known in the art. Compounds identified as being of therapeuticvalue may be subsequently analyzed in the 7B2 knockout mouse describedherein to determine if they can alleviate or exacerbate the symptoms ofthe diseased animal.

Administration of Reagents that Alter 7B2 Expression or Function

A 7B2 protein, a 7B2-encoding DNA, or a 7B2 expression- orfunction-altering compound may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form to patients suffering from an endocrine disorder.Administration may begin before or after the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intra-arterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intrathecal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration. Therapeutic formulations may be in the form ofliquid solutions or suspensions; for oral administration, formulationsmay be in the form of tablets or capsules; and for intranasalformulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences (18^(th) edition), ed.A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for 7B2 protein, 7B2 gene, or 7B2 expression- orfunction-enhancing compound compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

OTHER EMBODIMENTS

While the treatment regimens described herein are preferably applied tohuman patients, they also find use in the treatment of other animals,such as domestic pets or livestock.

Moreover, while the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims. All references are herein incorporated by reference.

1. A transgenic non-human mammal, wherein a gene encoding 7B2 protein ismodified, whereby 7B2 protein activity is reduced. 2-13. (canceled) 14.A nucleic acid vector comprising nucleic acid capable of undergoinghomologous recombination with an endogenous 7B2 gene in a cell, whereinsaid homologous recombination results in a modification of the 7B2 generesulting in decreased 7B2 protein activity in the cell.
 15. (canceled)16. A eukaryotic cell, wherein the endogenous gene encoding 7B2 proteinis modified, resulting in reduced 7B2 protein activity in the cell.17-18. (canceled)
 19. A eukaryotic cell containing an endogenous 7B2gene into which there is integrated a transposon comprising DNA encodinga selectable marker.
 20. A method for diagnosing a mammal for anendocrine disorder, said method comprising determining whether 7B2protein activity is abnormal in said mammal, whereby said abnormalactivity indicates that said mammal has an endocrine disorder or anincreased likelihood of developing an endocrine disorder. 21-34.(canceled)
 35. A method for determining whether a compound ispotentially useful for treating or alleviating the symptoms of anendocrine disorder, said method comprising: (a) providing a cellcomprising a reporter gene operably linked to the promoter from a 7B2gene; (b) contacting said cell with said compound; and (c) measuring theexpression of said reporter gene; whereby a change in the level of saidexpression in response to said compound indicates that the compound ispotentially useful for treating or alleviating the symptoms of anendocrine disorder.
 36. A method for determining whether a compound ispotentially useful for treating or alleviating the symptoms of anendocrine disorder, said method comprising: (a) providing a cell thatproduces a 7B2 protein; (b) contacting said cell with said compound; and(c) monitoring the activity of said 7B2 protein; whereby a change inactivity in response to said compound indicates that the compound ispotentially useful for treating or alleviating the symptoms of anendocrine disorder. 37-47. (canceled)